It is proposed to develop and exploit methodology which will permit synthesis of natural hemes regioselectively labeled in predetermined positions with deuterium, carbon- 13, tritium, nitrogen- 15, and fluorine, and these will be used as NMR and resonance Raman probes to gain an understanding of heme apoprotein interactions and structure/function relationships in a variety of biologically important heme proteins such as hemoglobins, myoglobins, cytochromes, and peroxidases. It is also proposed to synthesize a variety of unlabeled heme analogues, heme dimers, iron oxophlorins, and hydrdohemes. The proposed work will provide a basis for understanding heme protein function, and of structural and electronic factors which cause several debilitating diseases such as anemias. The synthetic compounds will also be used for EPR, X-ray, MCD, and other biological studies in collaboration with a number of independent investigators. After reconstruction into appropriate apoproteins, the novel synthetic compounds will be used to produce spectroscopic assignments, and for simplification of spectra and interpretations of a wide variety of heme proteins. Tow fundamental approaches for synthesis of novel compounds will be developed; carbon-13, nitrogen-15, fluorine labeled, and unlabeled prophyrins will be obtained by total synthesis from acyclic precursors. Methods for such approaches have already been worked out, but improvements and discovery of new methodology is planned and anticipated. Some deuterium labeled, carbon-13 labeled porphyrins, protoporphyrin IX analogues, oxophlorins, and sulfhemes, and other chlorin hemes will be approached by manipulation of substituents on existing commercially available porphyrins or from chlorophyll alpha which will be extracted form a commercially available alga. After method discovery and optimization, and in order to fully exploit the synthetic developments which usually take place only on limited scales, we plan to prepare most labeled, unlabeled and structurally modified hemes in batches of approximately 100 mg; these compounds will then be provided to collaborators so that biologically important multinuclear NMR, resonance Raman, EPR, X-ray, MCD, and other properties of the hemes, reconstituted heme proteins, non-iron metal complexes and structural modifications of the porphyrin ligands can be investigated, correlated, rationalized and interpreted.